Vaccination is an efficient way of preventing death or disability from infectious diseases. The success of this method in the field of infectious disease has also stimulated interest in utilizing vaccination in the treatment or prevention of neoplastic disease. Despite the successes achieved with the use of vaccines, however, there are still many challanges in the field of vaccine development. Parenteral routes of administration, the numbers of different vaccinations required and the need for, and frequency of, booster immunizations all impede efforts or eliminate disease.
One such difficulty is lack of immunogenicity, i.e., the antigen is unable to promote an effective immune response against the pathogen. In addition, certain antigens may elicit only a certain type of immune response, for example, a cell-mediated or a humoral response. Adjuvants are substances that enhance, augment or potentiate an immune response, and can in some instances, be used to promote one type of immune response over another. Although numerous vaccine adjuvants are known, aluminum salt is the only adjuvant widely used in humans, not, however, without any safety concern.
There is now convincing evidence that the immune system can recognize, and in some cases destroy, malignant cells and infectious agents. Furthermore, T cells, and in particular CD8+ cytotoxic T lymphocytes (CTLs), appear to be the principal affectors of anti-tumor and anti-infectious disease immunity. Activation of T cells is known to be dependent on dendritic cells. Dendritic cells (DC) are unique among antigen presenting cells (APC) by virtue of their potent capacity to activate immunologically naive T cells (Steinman, 1991). DC express constitutively, or after maturation, several molecules that mediate physical interaction with and deliver activation signals to responding T cells. These include class I and class II MHC molecules. CDSO (B7-1) and CD86 (B7-2); CD 40; CD11a/CD18 (LFA-1); and CD54 (ICAM-1) (Steinman, 1991; Steinman et al. 1995). The unique ability of dendritic cells to present antigens and to activate naive and memory CD4+ and CD8+ T cells provides the possibility of using them to trigger specific anti-tumor immunity. Therefore, an agent that could selectively induce dendritic cells and increase their ability to stimulate immune response would be of wide importance. Numerous studies have shown a high potency of dendritic cell-based vaccines for cancer immunotherapy in animal models, some have been carried out against human cancers in clinical trials. Human tumors express a number of protein antigens that can be recognized by cytotoxin lymphocytes (CTL), thus providing potential targets for cancer immunotherapy.
Dendritic cells (DCs) are rare leukocytes that are uniquely potent in their ability to present antigens to T cells, and this property has promoted their recent application to therapeutic cancer vaccines. Other cells are also known to be able to present antigens such as macrophages and B-cells. However, macrophages cannot take up soluble antigens efficiently, while immature dendritic cells can take up large amount of antigen from extracellular fluid by macropinocytosis.
B-cells, by contrast, are uniquely adapted to bind specific soluble molecules through their cell-surface immunoglobulin. B-cells internalize the soluble antigen bound by their immunoglobulin receptors and then display peptide fragments of these antigens as peptides: MHC class II complexes. The problem with B-cells is that they do not constitutively express co-stimulatory activity. Although B-cells efficiently present soluble proteins, they are unlikely to initiate a potent CTL response in the absence of co-stimulatory activity. As a result the antigen not only fails to activate naive T-cells, but causes them to become anergic, or non-responsive.
Isolated DCs loaded with tumor antigen ex vivo and administered as a cellular vaccine have been found to induce protective and therapeutic anti-tumor immunity in experimental animals. In pilot clinical trials of DC vaccination for patients with non-Hodgkin's lymphona and melanoma, induction of anti-tumor immune responses and tumor regressions have been observed. Timmerman et al., Annal, Rev Med 1999, 50:507-29; Tarte et al., Leukemia, 13:653-663 (1999). Additional trials of DC vaccination for a variety of human cancers are under way, and methods for targeting tumor antigens to DCs in vivo are also being explored. Exploitation of the antigen-presenting properties of DCs thus offers new possibilities for the development of effective cancer immunotherapies. Therefore, DCs can be used as a cell vaccine, but they can also be used as an immunomodulating factor in combination with DNA vaccine. Following DNA vaccination, DCs efficiently present vaccine-encoded antigens. Casares et al., J. Exp. Med., 186(9):1481-6 (1997). Plasmid DNA has an adjuvant effect that promotes DC maturation and migration to lymphoid tissue. However, only a very low number of DCs are usually transfected with a direct injection of plasmid DNA, and a very low number of DCs migrate to the site of injection. Lane et al., Immunology, 11:308-313 (1999). The expression of antigen by directly tranfected DCs become undectable after 2 weeks, but memory CD4+ T cell responses are maintained over 40 weeks, questioning the role of persistent antigen in maintaining CD4+ T cell memory. Bacterial DNA (CpG motifs) induces maturation of Langerhans cells and of immature bone-marrow-derived DCs. Bacterially-derived lipopolysaccharide (LPS) has long been known to be an activator for DCs. By triggering a Th1-type response, not only can inflammatory T cells be recruited to sites of infection in order to activate macrophages, but also they attract neutrophils to the infected area by secreting chemokines.
Co-delivery of the GM-CSF adjuvant and glycoprotein D antigen boosts immune response during plasmid DNA vaccination with naked DNA. Flo et al., Vaccine, 18(28): 3242-53 (2000). Gene delivery has been used to express cytokines (interleukin-12) through the use of plasmid DNA encoding cytokines with poly(α-4-aminobutylglycolic acid) complexes. Maheshwari et al., Mol. Ther., 2(2): 121-130. (2000). The tumor suppressor (antigen) p53 and interleukin12 (as well as TNF-α and IFNγ) have been administered via gene delivery in a gene delivery system named “LPD” to initiate cytokine response and inhibit tumor growth. Whitmore et al., Gene Ther., 6(11) 1867-75 (1999). Intravenous injection of plasmids encoding the human FLT-3 ligand increase the number of functional and natural killer cells (NK). He et al., Hum. Gene Ther., 11(4): 547-54 (2000). Several workers have used FLT-3 to boost gene expression during a retroviral-mediated gene therapy. Murray et al., Hum. Gene Ther., 10(11): 1743-52 (1999) and Goerner et al., Blood, 94(7): 2287-92 (1999). FLT-3, as well as GM-CSF, has been used to induce development of dendritic cells and boost gene expression during a retrovirus-mediated gene vaccination therapy. Mach et al., Cancer Res., 60(12): 3239-46 (2000). CD40 and FLT-3 ligands induce dendritic cells and boost gene expression during a retrovirus-mediated gene vaccination therapy. Borges et al., J. Immunol. 163(3) 1289-97 (1999).
The present invention relates to compositions comprising polynucleotides, such as plasmid DNA, DNA, RNA, viruses or vectors, and at least one block copolymer that induce an increased level of production and infiltration of DCs in response to the expression of the gene product encoded by the above DNA, in particular plasmid DNA. This event leads to a higher immune response against an encoded exogenous antigen (transgene), and a better humoral and cellular immune reponse is acheived. The compositions of the present invention can also be used to generate large amount of dendritic cells both in vitro and in vivo. The current methods of generation, stimulation, and maturation of DCs are extremely difficult and tedious, while the present invention significantly simplifies the process.
Direct injection of naked plasmid DNA either intramuscularly or intradermally induces strong, long-lived immune responses to the antigen encoded by the DNA vaccines. Both routes of immunization lead to production of specific antibodies and the activation of both MHC class I-restricted, antigen-specific CTL and MHC class II-restricted Th cells secreting Th1-type cytokines (Genetic vaccines, Scientific Amer., July 1999, pp. 50-57). These properties have made plasmid DNA vaccines an attractive alternative to conventional immunizations using proteins, live attenuated viruses or killed whole organisms. Consequently, DNA vaccines are actively being investigated as therapies or preventive measures in such diverse areas as infectious diseases, allergies, and cancers. Despite the avid interest in this method of immunization, DNA vaccines are limited by the capacity to express the protein. An efficient immunization is dependent upon gene expression, which means that the DNA vaccines have to express the protein.
The unique features of smooth, skeletal, and cardiac muscles, have presented numerous challenges for the development and administration of effective polynucleotide compositions for intramuscular administration. Direct injection of purified plasmids (“naked DNA”) in isotonic saline into muscle was found to result in DNA uptake and gene expression in smooth, skeletal, and cardiac muscles of various species. Rolland A., Critical Reviews in Therapeutic Drug Carrier Systems, Begell House, 143 (1998). It is believed that the unique cytoarchitectural features of muscle tissue are responsible for the uptake of polynucleotides because skeletal and cardiac muscle cells appear to be better suited to take-up and express injected foreign DNA vectors relative to other types of tissues. Dowty & Wolff, Gene Therapeutics: Methods and Applications of Direct Gene Transfer, Birkhäuser, Boston, p. 182 (1994). The relatively low expression levels attained by this method, however, have limited its applications. See Aihara and Miyazaki, Nature Biotechnology, 16:867 (1998). Additionally, traditional gene delivery systems such as polycations, cationic liposomes, and lipids that are commonly proposed to boost gene expression in other tissues usually result in inhibition of gene expression in skeletal and cardiac muscles. Dowty & Wolff, loc. cit., p. 82 (1994).
Even if the muscle is known to be the only tissue that efficiently takes up and expresses plasmid DNA in the absence of a viral vector, the muscle is not considered to be a site for antigen presentation because it contains few if any dendritic cells, macrophages, and lymphocytes. The skin and mucous membranes are the anatomical sites where most exogenous antigens are normally encountered. The skin-associated lymphoid tissue contain specialized cells that enhance immune responses. Raz et al., PNAS, 91: 9519-9523 (1994).
Anionic polymers such as dextran sulfate and salmon DNA can decrease gene expression in the muscle. Rolland A., Loc. cit. Various noncondensive interactive polymers have been used with a limited success to modify gene expression in striated muscle. Nonionic polymers such as poly(vinyl pyrrolidone) poly(vinyl alcohol) interact with plasmids through hydrogen bonding. Id. These polymers may facilitate the uptake of polynucleotides in muscle cells and cause up to 10-fold enhancement of gene expression. However, to achieve a significant increase in gene expression, high concentrations of polymers (about 5% and more) need to be administered. Mumper et al., Pharmacol. Res., 13, 701-709 (1996); March et al., Human Gene Therapy, 6(1), 41-53 (1995). This high concentration of poly(vinyl pyrrolidone) poly(vinyl alcohol) needed to improve gene expression can be associated with toxicity, inflammation, and other adverse effects in muscle tissues. Block copolymers have been used to improve gene expression in muscle or to modify the physiology of the muscle for subsequent therapeutic applications. See U.S. Pat. Nos. 5,552,309; 5,470,568; 5,605,687; and 5,824,322. For example, block copolymers can be used in a gel-like form (more than 1% of block copolymers) to formulate virus particles used to perform gene transfer in the vasculature. In the same range of block copolymers concentration (1-10%), it is possible with block copolymer to modify the permeability of damaged muscle tissue (radiation and electrical injury, and frost bite). In addition DNA molecules can be incorporated into cells following membrane permeabilization with block copolymers. For these applications, block copolymers were used at concentrations giving gel-like structures and viscous delivery systems. These systems are unlikely to enable diffusion of the DNA injected into the muscle, however, thus limiting infusion of the DNA into the myofibers.
There is thus a need for compositions and methods increasing efficacy of polynucleotides expression upon administration to a patient, in particular, in the muscle and in the skin. There is also a need for methods of increasing the efficiency of delivering polynucleotides to cells.
Beside the need to improve gene expression in muscle and skin, other tissues in the body would benefit from a gene transfer in a situation when there is a genetic disorder, and/or an abnormal over-expression of a gene, and/or absence of a normal gene.
Several polynucleotides such as RNA, DNA, viruses, and ribozymes can be used for gene therapy purposes. However, many problems, like the ones described below, have been encountered for successful gene therapies.
The use of antisense polynucleotides to treat genetic diseases, cell mutations (including cancer causing or enhancing mutations) and viral infections has gained widespread attention. This treatment tool is believed to operate, in one aspect, by binding to “sense” strands of mRNA encoding a protein believed to be involved in causing the disease site sought to be treated, thereby stopping or inhibiting the translation of the mRNA into the unwanted protein. In another aspect, genomic DNA is targeted for binding by the antisense polynucleotide (forming a triple helix), for instance, to inhibit transcription. See Helene, Anti-Cancer Drug Design, 6:569 (1991). Once the sequence of the mRNA sought to be bound is known, an antisense molecule can be designed that binds the sense strand by the Watson-Crick base-pairing rules, forming a duplex structure analogous to the DNA double helix. Gene Regulation: Biology of Antisense RNA and DNA, Erikson and lxzant, eds., Raven Press, New York, 1991; Helene, Anti-Cancer Drug Design, 6:569 (1991); Crooke, Anti-Cancer Drug Design, 6:609 (1991). A serious barrier to fully exploiting this technology is the problem of efficiently introducing into cells a sufficient number of antisense molecules to effectively interfere with the translation of the targeted mRNA or the function of DNA.